PROJECT SUMMARY T cell activation requires a cascade of T cell receptor signaling events including the Ca2+-NFAT pathway. CRAC (Ca2+ release-activated Ca2+) channels are pivotal for activation of this pathway by elevating intracellular Ca2+ concentrations. Defects in the Ca2+-NFAT pathway lead to immune deficiency in humans and blockers for this pathway including cyclosporin A and FK506 are widely used to suppress T cell functions in the clinic. The major problem with therapeutic exploitation of the Ca2+-NFAT pathway is that inhibition of this pathway leads to systemic immune suppression due to its broad role in most T cell functions. Our goal is to elucidate mechansims regulating CRAC channels specifically in local effector T cells and develop a therapeutic method to suppress T cell responses in inflamed tissues. Stimulation of T cell receptors (TCRs) induces depletion of endoplasmic reticulum Ca2+ stores. An endoplasmic reticulum-resident signaling molecule, STIM1, senses this depletion and interacts with Orai1, the pore subunit of CRAC channels to induce Ca2+ entry. Our research within the last decade using proteomics combined with affinity protein purification has successfully identified several regulators that play important roles in Orai1-STIM1 interaction in T cells. New preliminary data now reveal a novel vesicle trafficking-mediated mechanism in regulation of CRAC channels in effector T cells. From a targeted screen using an expression library of EF-hand containing proteins, we have identified a vesicular component, NKD2 (naked cuticle homolog 2) as an important regulator of CRAC channels. Mechanistically, NKD2 co-localizes with intracellular vesicles containing Orai1, and upon TCR stimulation, mediates trafficking of this intracellular vesicular Orai1 to the plasma membrane. Furthermore, NKD2 is abundantly expressed in effector T cells compared to other cell types and its deletion specifically decreased local effector T cell functions without influencing T cell functions in the lymphoid organs. Taken together, these results lead to the main hypothesis of the current proposal that trafficking of intracellular Orai1 vesicles is crucial for high and sustained levels of Ca2+ signaling required for local effector T cell responses. Identification of NKD2 provides a molecular handle to understand this unique mechanism. The objective of this proposal is to determine 1) the upstream TCR signals that activate NKD2 to induce Orai1 trafficking, 2) downstream effector molecules (e.g., Rab GTPases) that mediate trafficking of NKD2/Orai1 vesicles, and 3) the cellular mechanisms underlying the reduced autoimmune symptoms observed in NKD2 knockout mice.